Mechanism of Chalcone Synthase

Polyketide synthases (PKS) assemble structurally diverse natural products using a common mechanistic strategy that relies on a cysteine residue to anchor the polyketide during a series of decarboxylative condensation reactions that build the final reaction product. Crystallographic and functional studies of chalcone synthase (CHS), a plant-specific PKS, indicate that a cysteine-histidine pair (Cys164-His303) forms part of the catalytic machinery. Thiol-specific inactivation and the pH dependence of the malonyl-CoA decarboxylation reaction were used to evaluate the potential interaction between these two residues. Inactivation of CHS by iodoacetamide and iodoacetic acid targets Cys164 in a pH-dependent manner (pK a = 5.50). The acidic pK a of Cys164 suggests that an ionic interaction with His303 stabilizes the thiolate anion. Consistent with this assertion, substitution of a glutamine for His303 maintains catalytic activity but shifts the pK a of the thiol to 6.61. Although the H303A mutant was catalytically inactive, the pH-dependent incorporation of [14C]iodoacetamide into this mutant exhibits a pK a = 7.62. Subsequent analysis of the pH dependence of the malonyl-CoA decarboxylation reaction catalyzed by wild-type CHS and the H303Q and C164A mutants also supports the presence of an ion pair at the CHS active site. Structural and sequence conservation of a cysteine-histidine pair in the active sites of other PKS implies that a thiolate-imidazolium ion pair plays a central role in polyketide biosynthesis.

Polyketide synthases (PKS) 1 from bacteria, fungi, and plants produce an array of natural products (1)(2)(3)(4). Many polyketides possess pharmacological properties and are used as antibiotics, immunosuppressants, anti-cancer agents, and anti-fungal agents (5)(6). Despite the structural complexity of these compounds, a common chemical strategy underlies the biosynthetic mechanisms of different PKS. The initial reaction step involves loading a starter molecule onto an active site cysteine through an acyltransferase activity. Following formation of the primed acyl-enzyme complex, a decarboxylative condensation reaction extends the reaction intermediate. The elongation step can be repeated until an appropriate chain length is reached and the reaction product released. This process is analogous to the reactions catalyzed by fatty acid synthases (FAS) (7). Recent structural and kinetic studies of chalcone synthase (CHS), a plant-specific PKS, have elucidated the basis of polyketide formation in plants and provide a model for understanding the reaction mechanism of other PKS.
Unlike the modular PKS, such as 6-deoxyerythronolide B synthase, which are large protein assemblies with distinct active sites that catalyze each elongation step (1-2, 4, 6), the plant-specific PKS function as homodimeric iterative PKS (monomer, molecular mass ϳ 42 kDa) that perform consecutive elongation reactions at two independent active sites (3,8). CHS uses p-coumaroyl-CoA as a starter molecule and three malonyl-CoA extender molecules to form a tetraketide intermediate that is cyclized into 4,2Ј,4Ј,6Ј-tetrahydroxychalcone (chalcone) (Fig. 1a) (9). This activity is central to the biosynthesis of anti-microbial isoflavonoid phytoalexins, anthocyanin floral pigments, and flavonoid inducers of Rhizobium nodulation genes (10,11). Also, flavonoids are of interest as pharmacological agents (12)(13)(14) and are constituents in plant-rich diets associated with a reduced risk of cardiovascular disease and some forms of cancer (15,16).
The three-dimensional structure of alfalfa CHS2 provides a view of the active site that catalyzes chalcone formation ( Fig.  1b) (17). Four residues (Cys 164 , His 303 , Asn 336 , and Phe 215 ) form the catalytic center of CHS and are strictly conserved in other CHS-like enzymes, including 2-pyrone synthase (18), stilbene synthase (19), bibenzyl synthase (20), acridone synthase (21), and the rppA CHS-like protein (22). Based upon structural and functional studies of CHS (17,23,24), the proposed reaction mechanism involves Cys 164 acting as the nucleophilic thiolate in the loading reaction and as the covalent thioesteranchor for the acyl-enzyme chain during the elongation reactions (Fig. 1c). In addition, His 303 and Asn 336 catalyze the decarboxylation of malonyl-CoA in the elongation reaction and stabilize the transition state during the condensation phases of polyketide formation. Phe 215 may orient substrates and reaction intermediates at the active site.
In the crystal structure of CHS (17), the S␥ of Cys 164 forms a hydrogen bond with the N⑀ of His 303 , which is 3.5 Å distant (Fig. 1b). Previous mutagenesis studies suggest that His 303 does not act as a general base by abstracting a proton from Cys 164 to form the reactive thiolate required for chalcone formation (24). Rather, at physiological pH, these two residues may form a stable imidazolium-thiolate ion pair. In other enzymes, notably the cysteine proteases (25-28), the thiolate anion of the catalytic cysteine is stabilized by an imidazolium * This work was supported by a grant from the National Science Foundation (MCB9982586 to J. P. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ A National Institutes of Health postdoctoral research fellow (CA80396) and also the recipient of support from the Hoffman Founda- ion on an adjacent histidine. The structural proximity of Cys 164 and His 303 in the CHS active site raises the potential for a similar mechanistic feature in this PKS. This paper describes the use of thiol-specific inactivators to evaluate the reactivity of Cys 164 , to determine the pK a of the active site cysteine, and to establish the role of His 303 in maintaining the reactivity of Cys 164 . In addition, the pH dependence of the malonyl-CoA decarboxylation reactions catalyzed by wild-type CHS and the H303Q and C164A mutants was examined. These studies demonstrate that Cys 164 is a reactive thiolate anion with an acidic pK a that is modulated by interaction with His 303 . Combined with previous crystallographic and kinetic studies, this work provides insight on the mechanism of CHS and suggests that a thiolate-imidazolium ion pair plays a significant role in both polyketide and fatty acid biosynthesis.  (29) with reagents purchased from Aldrich or Sigma. Electrospray mass spectroscopy to confirm the identity of the synthesis product was performed by the Mass Spectrometry Facility of the Scripps Research Institute.

Materials-[2-
Preparation of Wild-type and Mutant CHSs-The CHS C164A, H303Q, and H303A mutants were generated previously (24). Wild-type and mutant CHS proteins were expressed and purified to homogeneity as described elsewhere (24).
CHS Assay-The standard assay for determining CHS activity was conducted in 100-l systems containing 100 mM potassium phosphate buffer (pH 7.0), 50 M [2-14 C]malonyl-CoA (50,000 cpm), and 15 M coumaroyl-CoA at 25°C (24). Reactions were initiated by the addition of enzyme and were quenched by ethyl acetate extraction. Extracts were evaporated to dryness and re-dissolved in methanol. Scintillation counting to detect radioactivity in the methanol sample was performed in Ecolume scintillation fluid.
Inactivation Studies-Wild-type CHS (10 g) was incubated in 30-l volumes using a triple buffer system (30, 31) (50 mM AMPSO, 50 mM sodium phosphate, and 50 mM sodium pyrophosphate, pH 7.0) in the presence of 0 -50 M iodoacetamide or iodoacetic acid at 25°C. Similar experiments with the H303Q mutant (10 g) were performed in the presence of 0 -500 M iodoacetamide or 0 -1000 M iodoacetic acid under the same reaction conditions. All reactions were initiated by the addition of inactivator. Aliquots (2 l) were withdrawn from the incubation mixture and diluted into the standard assay system, and the amount of enzyme activity remaining determined. All inactivation experiments were monitored relative to a control sample without inactivator, which is set to 100% activity at each time point. This control accounts for the loss of enzyme activity under experimental conditions. Inactivation data were plotted as log (% initial enzyme activity) versus time. Semi-log plots were fitted to the first-order equation, ϪdE/dt ϭ k(I), where it is assumed that the disappearance of enzyme activity over time is related to the concentration of either iodoacetamide or iodoacetic acid, (I), multiplied by k, a rate constant. This allowed determination of the half-life for inactivation (t1 ⁄2 ) at each inactivator concentration. Kitz-Wilson analysis of the data was used to generate the limiting constant for inactivation (k inact ) and K I by plotting t1 ⁄2 versus 1/(inactivator, mM) (32).
Determination of Active Site Labeling-Wild-type CHS (10 g), H303Q mutant (10 g), or C164A mutant (25 g) were incubated with either 5 M [2-14 C]iodoacetamide or 10 M [1-14 C]iodoacetic acid at 25°C in the triple buffer system (pH 7.0) as described above. Two aliquots were removed at each time point. One sample was assayed for CHS activity. The second sample was diluted 50-fold with triple buffer and loaded onto a G-25 Sephadex Quick-spin column. The radiolabeled FIG. 1. CHS reaction and active site structure. a, overall CHS reaction; b, CHS active site. The catalytic cysteine and conserved histidine are shown along with the terminal end of a CoA molecule and adjacent residues that have had their catalytic roles investigated previously. The hydrogen bond between Cys 164 and His 303 is indicated by the dotted line. This view is oriented looking down the pantethine arm of the CoA. c, proposed reaction mechanism for CHS. Loading, decarboxylation, and elongation steps are shown. R is the coumaroyl moiety in the first reaction cycle, coumaroyl-acetyl group in the second cycle, and a coumaroyl-diacetyl group in the third cycle. The proposed cysteine-histidine ionic interaction in the loading is indicated.
protein in the eluant was quantitated by scintillation counting in Ecolume. Total protein concentration in the flow-through was determined spectrophotometrically (A 280 nm ; ⑀ ϭ 37,680 M Ϫ1 cm Ϫ1 /monomer). Controls in which either [ 14 C]iodoacetamide or [ 14 C]iodoacetic acid was loaded on a spin column showed that no significant amount of radioactivity was in the flow-through.
pH Dependence of Inactivation and Active Site Labeling of CHS and the His 303 Mutants-Wild-type CHS (10 g) or the H303Q mutant (10 g) were incubated in the triple buffer system at the specified pH values (4.62-8.27) in the presence of varying concentrations of iodoacetamide over a range of times, as described above. The triple buffer system maintains a constant ionic strength across the pH range used (30 -31). Aliquots were removed and assayed using the standard CHS assay. From these incubations and assays, the second-order rate constant (k 2 ) at each pH was determined for both enzymes. The pH profiles (log(k 2 ) versus pH) were fitted as described by Cleland (33) using Kaleidagraph (Abelbeck Software).
To determine the pH dependence of iodoacetamide labeling of the H303A mutant, purified protein (25 g) was incubated with [ 14 C]iodoacetamide at 25°C in the triple buffer system (pH range, 4.62-9.02) (30-l reaction volume). Incubations were conducted at 50 and 100 M for 5, 15, 30, and 60 min to obtain the rates of incorporation of [ 14 C]iodoacetamide at each pH. At the specified times, each sample was diluted 50-fold with triple buffer at the same pH, loaded onto a G-25 Sephadex Quick-spin column, and then eluted. Radiolabeled protein in the eluant was quantitated by scintillation counting in Ecolume. Incorporation of [ 14 C]iodoacetamide in the H303A mutant was linear over the time course at both concentrations and depleted a maximum of 5% of the available enzyme. The pH profile was fit as above.
pH Dependence of Malonyl-CoA Decarboxylation-Determination of the k cat and K m values for malonyl-CoA decarboxylation catalyzed by wild-type CHS (0.5 g) and the H303Q (1 g) and C164A (1 g) mutants used a radiometric assay that monitors conversion of [ 14 C]malonyl-CoA to [ 14 C]-acetyl-CoA (24). Assays used the triple buffer system (pH range, 5.04 -8.50) and varied malonyl-CoA concentrations (2.2-220 M; 10,000 -50,000 cpm) in a 50-l reaction volume at 25°C. Data was obtained from initial velocity experiments in which product formation was linear over the time periods monitored. Kinetic constants were calculated by nonlinear least squares iterative curve fitting to the Michaelis-Menton equation using Kaleidagraph. The pH profiles were fit as described above.

Kinetics of CHS Inactivation by Iodoacetamide and Iodoace-
tic Acid-Affinity-labeling agents and enzyme inactivators provide useful probes of active site chemistry in enzymes. Thiolspecific compounds, such as iodoacetamide and iodoacetic acid, capitalize on the ability of a sulfhydryl group to be sufficiently nucleophilic to rapidly react in solution. Early work on CHS from cell suspension cultures qualitatively demonstrated that iodoacetamide and iodoacetic acid inactivated the enzyme (9,34). Using homogenous recombinant protein, the inactivation of CHS by iodoacetamide and iodoacetic acid was re-examined. CHS is inactivated by both compounds in a pseudo-first-order kinetic manner (Fig. 2, a and c). Inactivation of CHS by iodoacetamide and iodoacetic acid displayed second-order rate constants (k 2 ) of 1390 and 635 M Ϫ1 s Ϫ1 , respectively, as determined by Kitz-Wilson analysis (Fig. 2, b and d).
Identification of Inactivator Attachment Site-The CHS active site contains one cysteine residue (Cys 164 ) (17,24). To confirm that Cys 164 is specifically targeted by iodoacetamide and iodoacetic acid, purified wild-type CHS and the C164A mutant were incubated with [ 14 C]iodoacetamide and [ 14 C]iodoacetic acid. Incorporation of radioactive iodoacetamide into wild-type CHS coincided with a loss of activity for chalcone formation (Fig. 3). Similar results were obtained when wildtype CHS was incubated with radioactive iodoacetic acid (not shown). To ensure that modification of the active site thiol was covalent, wild-type CHS and inactivated wild-type CHS were dialyzed against reaction buffer and re-assayed for CHS activity. After dialysis, enzyme treated with iodoacetamide or iodoacetic acid lacked activity, whereas untreated wild-type enzyme retained catalytic activity. Stoichiometries of 0.80 mol [ 14 C]iodoacetamide/mol of CHS monomer and 0.68 mol [ 14 C]iodoacetic acid/mol of CHS monomer were determined from the amount of radiolabeled inactivator incorporated into the enzyme. Because CHS is a homodimeric protein with two active sites that are catalytically independent (7), the stoichiometries suggest that both inactivators target the active site cysteine of each monomer. Incubation of the C164A mutant with [ 14 C]iodoacetamide (Fig. 3) or [ 14 C]iodoacetic acid (not shown) under identical reaction conditions as wild-type CHS showed that negligible amounts of radioactivity were incorporated into the mutant protein. Therefore, Cys 164 is the specific residue targeted by both iodoacetamide and iodoacetic acid that results in loss of CHS activity.
The pK a of the Catalytic Cysteine-In the acyltransferase reaction that loads the starter molecule onto CHS, a thiolate anion is required as the nucleophile. However, physiological pH is 1 unit below the accepted pK a value range of 8.0 -8.5 for a cysteine sulfhydryl (35). Therefore, the pK a of the catalytic cysteine in CHS must shift below 7.0 to serve as an effective nucleophile. To determine the pK a of Cys 164 , the pH dependence of inactivation was evaluated using iodoacetamide. The second-order rate constants (k 2 ) for CHS inactivation by iodoacetamide were determined over a pH range from 4.62 to 8.27. Wild-type CHS inactivated with a pK a of 5.50 Ϯ 0.10 and exhibited a 1 log-unit change in the value of k 2 over a single pH unit (Fig. 4), consistent with the titration of a single ionizable group (36).
Effect of the Conserved Histidine on Inactivation Kinetics-Inactivation of the CHS H303Q mutant by iodoacetamide and iodoacetic acid followed pseudo-first-order kinetics (Fig. 5, a  and c) and displayed second-order rate constants (k 2 ) of 275 and 30.3 M Ϫ1 s Ϫ1 , respectively (Fig. 5, b and d). Comparison of the second-order rate constants of the H303Q mutant with wild-type enzyme indicates that the reactivity of the active site thiol is reduced. The incorporation of radiolabeled iodoacetamide and iodoacetic acid into the H303Q mutant was covalent, coincided with loss of enzymatic activity, and yielded stoichiometries of 0.85 and 0.71 mol inactivator/mol H303Q monomer, respectively (not shown). Importantly, the pH dependence of iodoacetamide inactivation of the H303Q mutant revealed a pK a ϭ 6.61 Ϯ 0.12 (Fig. 4). Relative to wild-type CHS, the difference in the pK a of inactivation and the reactivity of the thiol in the H303Q mutant is consistent with the proposal that a positively charged His 303 plays a role in stabilizing the thiolate of Cys 164 and maintaining its reactivity.
As described previously (24), the H303A mutant lacked catalytic activity, and therefore, inactivation experiments to evaluate the pK a of Cys 164 in this protein could not be performed. However, using [ 14 C]iodoacetamide, the pK a of iodoacetamide labeling of the H303A mutant was determined. An affinity label (R) forms a reversible complex prior to irreversible cova-lent modification (E ϩ R 7 ER 3 EX) that follows the equation k obs ϭ k max /(1 ϩ (K I /(R))), where k obs is the observed rate constant for modification, k max is the maximal rate for modification at saturating concentrations of R, and K I represents the concentration of reagent giving half-maximal inactivation rate. When (R)Ͻ ϽK I , k obs equals the pseudo-bimolecular rate constant k max /K I , which has units of M Ϫ1 min Ϫ1 . Initially, incubation of the H303A mutant with up to 1 mM radiolabeled iodoacetamide did not exhibit saturation kinetics (not shown). Therefore, lower concentrations of [ 14 C]iodoacetamide were used to determine the pseudo-bimolecular rate constant over a range of pH values (Fig. 6). For comparison with the secondorder rate constants for inactivation of wild-type CHS and the H303Q mutant at pH 7.0, the k max /K I for [ 14 C]iodoacetamide labeling of the H303A mutant was 0.090 M Ϫ1 s Ϫ1 . The pH dependence of k max /K I for iodoacetamide inactivation of the H303A mutant revealed a pK a ϭ 7.62 Ϯ 0.09 (Fig. 6).
Since the pH range used may include other reactive cysteines, the C164A mutant was incubated with [ 14 C]iodoacetamide under the same conditions as the H303A mutant. In this control experiment, there was no significant radioactivity detected in the C164A mutant protein until pH 8.5-9.0 (not shown) was reached. At these basic pH values, the amount of radioactivity was less than 10% of the total measured with the H303A mutant. This result indicates that the pH profile determined for the H303A mutant results from titration of Cys 164 .
Influence of Cys 164 and His 303 on the pH Dependence of Malonyl-CoA Decarboxylation-To assess the catalytic role of the thiolate-imidazolium ion pair at the CHS active site, the pH dependence of k cat and k cat /K m for malonyl-CoA decarboxylation catalyzed by wild-type CHS and the H303Q and C164A mutants was determined over a pH range of 5.04 to 8.50 (Fig.  7). Because of the multiple catalytic steps involved in chalcone formation, each requiring different protonation states of CHS or the substrates and intermediates, the malonyl-CoA decarboxylation reaction was examined to simplify interpretation of these experiments. The pH dependence of k cat with wild-type CHS displays a wave-shaped profile that levels off at both high and low pH with a pK a ϭ 6.94 Ϯ 0.09. In both the H303Q and C164A mutants, this break point is eliminated. The k cat profile for wild-type CHS reveals that the enzyme-substrate complex required for malonyl-CoA decarboxylation undergoes a change in protonation state, which does not occur when either His 303 or Cys 164 is mutated to aprotic residues; this suggests that the rate-limiting step in the decarboxylation reaction is altered in each mutant. The log(k cat /K m ) versus pH profiles for malonyl-CoA decarboxylation catalyzed by wild-type CHS, the H303Q mutant, and the C164A mutant each exhibited a similar break point in the basic region with extrapolated pK a values of 9.23 Ϯ 0.73, 9.46 Ϯ 1.99, and 9.32 Ϯ 1.68, respectively. The presence of this break point in the k cat /K m profiles, and not the k cat profile, Loss of CHS activity in wild-type enzyme was monitored (ovals) together with incorporation of [ 14 C]iodoacetamide into the protein (squares). The amount of radioactivity incorporated into the C164A mutant under the same reaction conditions is shown (triangles). Reactions were performed in duplicate at pH 7.0 and 25°C in a triple buffer system as described under "Experimental Procedures. "   FIG. 4. pH dependence of CHS inactivation by iodoacetamide for wild-type CHS (ovals) and the H303Q mutant (squares). Conditions were as described under "Experimental Procedures." of each protein suggests that a basic residue is involved in substrate binding. This inflection point may represent any of the three arginine or lysine residues on the surface of CHS that interact with the phosphate moieties of CoA substrates (17,24). DISCUSSION Iodoacetamide and iodoacetic acid inactivate CHS through specific modification of Cys 164 at the active site. This result agrees with mutagenesis experiments of CHS in which substitution of the cysteine with a serine or alanine results in a complete loss of chalcone formation (23)(24). Importantly, the pK a of Cys 164 indicates that a thiolate anion is present at the CHS active site at physiological pH to serve as the nucleophile in the loading reaction and as the attachment site of the polyketide during the elongation reactions. The acidic pK a of Cys 164 also explains why the sulfhydryl group of this residue is oxidized to sulfinic acid (Cys-SO 2 H) in several crystal structures of CHS (17,24). Similar oxidation of the active site thiol in the cysteine proteases also occurs (37,38). In these enzymes, the reactive thiolate forms an ion pair with the imidazolium ion of an adjacent histidine residue (27). Although the pK a value determined for Cys 164 in CHS is not as low as the 3.3-4.0 pK a of the thiolate present at the active site of the cysteine proteases (25)(26)(27), the observed pK a of the CHS active site thiol is significantly shifted from the accepted pK a value of 8.0 -8.5 for a cysteine sulfhydryl moiety (35). A reduction in pK a of this magnitude requires stabilization by the local environment. The proximity of His 303 to Cys 164 in the CHS structure suggests that the histidine, as an imidazolium cation, stabilizes the thiolate anion (Fig. 8).
Consistent with the presence of an ion pair at the CHS active site, the reactivity and pK a of Cys 164 shifted when mutations of His 303 were made. Substitution of a glutamine for His 303 also maintains the thiolate of Cys 164 , but the resulting nucleophile is less reactive than in wild-type CHS. In the three-dimensional structure of the H303Q mutant (24), substitution of a glutamine for His 303 is isosteric with the amide nitrogen of Gln 303 hydrogen bonding the sulfhydryl of Cys 164 . Hydrogen bonding and a partial positive charge on the amide nitrogen arising from resonance stabilization promote formation of the thiolate at Cys 164 , albeit less efficiently and with a corresponding shift in pK a (Fig. 8).
No stabilizing effect would be expected from the side chain of Ala 303 in the H303A mutant (Fig. 8). As described previously (24), the lack of malonyl-CoA decarboxylation and chalcone formation activities in this mutant underscores the mechanis- tic importance of the histidine. Although the pK a for iodoacetamide labeling of the H303A mutant is shifted to 7.62, the observed pK a is still 0.4 -0.9 pH units below that of a free cysteine. Asn 336 is another polar residue near Cys 164 (4.1 Å), but substitution of the asparagine with an alanine does not alter the inactivation kinetics of the N336A mutant or the pK a of Cys 164 in this mutant. 2 Since no other direct interactions occur with Cys 164 , the local environment may alter the pK a of this residue. Cys 164 is at the N terminus of ␣-helix 9 in the CHS structure and the helix-dipole may further reduce the thiol's pK a in the absence of other interactions (40 -42).
If Cys 164 and His 303 form a stable ion pair, then the ionization states of both residues should be thermodynamically linked and a second inflection point observed in the pH profiles for inactivation. For example, in papain the pK a of the active site histidine shifts from 4.3 to 8.5 when the active site thiol is deprotonated (25)(26). Since the structure of wild-type CHS indicates that interaction between Cys 164 and His 303 occurs (17,24), it is likely that the absent inflection point is beyond the pH range studied. Therefore, the potential pK a of His 303 in the free enzyme must be greater than 9.0, which is also consistent with the activity of the H303Q mutant, in which the glutamine's amide pK a is approximately 17.0 (39). Under physiological conditions, a stable thiolate-imidazolium ion pair in the CHS active site would maintain the nucleophilic thiolate required for the loading reaction.
The pH dependence of the malonyl-CoA decarboxylation reaction also supports the importance of a charged interaction at the CHS active site. In wild-type enzyme, the presence of an imidazolium ion would enhance formation of an enolate anion in the decarboxylation reaction (43). Since the k cat pH profile corresponds to the protonation state of the enzyme-substrate complex, the assignment of the observed pK a to any particular residue is problematic. Most likely, the observed break point represents enolization of malonyl-CoA during the decarboxylation reaction. Mutation of either His 303 or Cys 164 to an aprotic side chain eliminates the observed inflection point, suggesting that the rate-determining step in the decarboxylation reaction is altered. Although the H303Q and C164A mutants retain hydrogen bond donors at the active site as a glutamine and a histidine, respectively, the ionic pair at the active site is disrupted. Loss of the imidazolium ion in these mutants would slow substrate enolization and eliminate the observed pH dependence on the decarboxylation reaction.
Since the active site residues of CHS are conserved among CHS-like PKS, including 2-pyrone synthase (18), stilbene synthase (19), bibenzyl synthase (20), acridone synthase (21), and the rppA CHS-like protein (22), these enzymes likely retain a thiolate-imidazolium ion pair at their active sites. In addition, homology of the CHS active site residues with those of the FAS and other PKS (44), such as 6-deoxyerythronolide B synthase and actinorhodin synthase, suggests that a similar ion pair may be a defining feature in these enzymes.
Functional studies of FAS II and III demonstrate that the role of the catalytic cysteine in these enzymes is identical to that of Cys 164 in CHS (45,46). Also, the rapid inactivation of FAS by iodoacetamide implies that the active site thiol is highly reactive (47)(48). Although studies on FAS III demonstrate the importance of the active site histidine in the overall reaction mechanism (42,46), the effect of this residue on the pK a of the catalytic cysteine in FAS has not been evaluated. Currently, no detailed structural information is available on the modular or heterodimeric iterative PKS, but mechanistic analysis of actinorhodin synthase suggests that a cysteinehistidine dyad is an essential catalytic component (49). In the reaction mechanism of actinorhodin synthase, a cysteine (Cys 169 ) serves as the attachment point for the polyketide chain (49 -51), and a histidine (His 346 ) may activate the thiol in the loading and elongation reactions.
Stabilization of negatively charged thiolates by imidazolium ions at the active sites of PKS eliminates the need for formal proton transfers such as those governed by general acid-base catalysis. Intuitively, the catalytic advantage of this mechanistic model may derive from limiting bond making and bond breaking steps to thioester formation and breakdown without additional proton transfers to and from the thiolate and imidazolium ions and their respective acids and bases.